Competitive binding immunoassays, which take advantage of natural immunological reactions, have found widespread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes which are present in very low concentration and cannot be adequately quantitated by chemical techniques. Such analytes include, for example, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding assays, a labeled analyte is placed in competition with unlabeled analyte for reaction with a fixed amount of the appropriate antibody. Unknown concentrations of the analyte can be determined from the measured signal of either the bound or unbound (i.e. free) labeled analyte.
Phenytoin and phenobarbital are two drugs which are of interest today in the treatment of seizure disorders, and accurate measurement of these drugs in body fluids is important. Generally, assays for these drugs are competitive binding immunossays involving the use of a labeled analog of the drug. That is, in an assay for phenytoin, a labeled analog of phenytoin is generally placed in competition with phenytoin for a fixed amount of antibodies to phenytoin. In an assay for phenobarbital, a labeled analog of phenobarbital is generally placed in competition with the phenobarbital for a fixed amount of antibodies to phenobarbital.
It was observed, however, that under some conditions, close structural analogs of the drugs produce assays which exhibit insufficient sensitivity or dynamic range for relatively low concentrations of phenytoin or phenobarbital.